A single nucleotide mutation drastically increases the expression of tumor-homing NGR-TNF[alpha] in the E. coli M15-pQE30 system by improving gene transcription

• Published in: Applied microbiology and biotechnology Vol. 105; no. 4; pp. 1447 - 1460
• Main Authors: Chen, Jie, Yang, Hao, Feng, Yanru, Shi, Qiuxiao, Li, Zhao, Tao, Ze, Fan, Jie, Jin, Youmei, Li, Shengfu, Cheng, Jingqiu, Lu, Xiaofeng
• Format: Journal Article
• Language: English
• Published: Springer 01.02.2021
• Subjects: Escherichia coli; Gene expression; Genetic aspects; Mutation (Biology); Physiological aspects; Tumor necrosis factor; China
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-021-11136-x

Due to their potent immune stimulation, tumor necrosis factor alpha (TNF[alpha]) variants with tumor-homing activity are attractive as novel antitumor drugs. The promising antitumor effect of NGR-TNF[alpha] in clinical trials triggered extensive interest in developing novel tumor-homing TNF[alpha] variants in recent years. Owing to its promising antitumor effect, NGR-TNF[alpha] is usually used as a control for newly developed tumor-homing TNF[alpha] variants. In our previous works, we produced a pericyte-targeting Z-TNF[alpha] at high levels using the Escherichia coli (E. coli) M15-pQE30 system. To further compare Z-TNF[alpha] and NGR-TNF[alpha], we attempted to express NGR-TNF[alpha] using the same system. Surprisingly, native NGR-TNF[alpha] was expressed at a low (~ 0.2 mg/L) level in E. coli M15 containing the pQE30 plasmid. However, a single nucleotide mutation of C to G, resulting in a substitution of leucine (L) with valine (V) at the start of TNF[alpha], increased the expression of NGR-TNF[alpha] by ~ 100 times through improving transcription. In addition, the amino acid substitution showed a little impact on the receptor binding, in vitro cytotoxicity, and in vivo antitumor effect of NGR-TNF[alpha]. As fusing NGR to the N-terminus of TNF[alpha] with a valine substitution did not reduce the protein yield, the TNF[alpha] gene with a C > G mutation might be used to prepare novel tumor-homing TNF[alpha] when the native TNF[alpha]-based variant is expressed at an extremely low level in E. coli. Notably, in addition to the mutated valine, the impact of N-terminal additional amino acids provided by pQE30 vector on the function of TNF[alpha] variant must be carefully evaluated.